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Targeted disruption of the mouse endothelin-1 gene
S6-A-1-01 ENDOTHELIN OF MESANGIAL ORIGIN GLOMERULAR MESANGIAL CELLS M.Kohno, T.Horio, E.Uchida, T.Takeda
K.Yokokawa,
MODULATES THE
K.Yasunari,
K.Murakawa,
MITOGENIC
H.Kano,
EFFECT
M.Ikeda,
OF PDGF
M.Minami,
ON
T.Hanehira,
First Department of Internal Medicine, Osaka City University Medical School, Osaka 545, JAPAN We have previously shown that cultured rat mesangial cells secrete endothelin-1 and this secretion is stimulated by PDGF (Am J Physiol 266:E673,1993). Here, we examined whether PDGF stimulate mesangial cell proliferation through the modulation of the endogenous production of endothelin-1 in these cells. Endothelin-1 level was measured by radioimmunoassay. Mitogenesis experiments were assessed by tritiated thymidine incorporation into DNA under highest concentrations (10ng/ml) of PDGF in the presence and absence of antiendothelin-1 antiserum or a selective A-type endotbelin receptor (ETA receptor) antagonist, BQ-123. PDGF AB and BB significantly stimulated mesangial cell production of endothelin-1, but PDGF AA did not affect endothelin-1 production. PDGF AB and BB potently stimulate thymidine incorporation, but PDGF AA did not affect thymidine incorporation. This stimulation was significantly attenuated in the presence of either anti-endothelin-1 antiserum or BQ-123 (10-7-10 -5 M). PDGF AB and BB significantly stimulate cell number and this stimulation was also attenuated in the presence of either anti-endothelin-1 antiserum or BQ-123. Anti-endothelin-1 antiserum and the highest concentration (10-SM) of BQ-123 did not affect basal thymidine incorporation and cell number significantly. Results suggest that PDGF AB and BB stimulate endothelin-1 secretion in rat mesangial cells through PDGF t3receptors, and endogenously produced endothelin-1 serves to modulate the effect of PDGF AB and BB, probably via ETA receptors in these cells. These data raise the possibility of the renoprotective effect of ETA receptor blocking in a certain renal disease.
$6-A-1-02 TARGETED DISRUPTIONOF THE MOUSEENDOTHELIN-1GENE I-L Knrihar#, Y. Knrihar#, K SuzukPa, T. Kodama~, K. Maemnra1,1~ NagaP, K Oda', T. KuwakP, T. Ishikawa~, M. Kumada', Y. YazakiI 1)The Third Department of Internal Medicine,3)Department of Pathology, 4)Department of Physiology, University of Tokyo, Tokyo, Japan 2)Chugai Pharmaceutical Co. Ltd., Shiznoka, Japan To elucidate the pathophysiological rain of endothelin-1 (ET-1), we established ET-1 knockout mice by gene targeting and analyzed the phenotype of homozygotes and hetero~ygotes. We introduced the targeting vector constructed from 7.5 kb mouse ET- 1 genornicDNA fragment into A3.1 ES cells by electtopomfion, and obtained 35 targeted ES clones using positive-negative selection. One of the targeted clones gave two chimeric mice with germ-line transmission and we obtained ET-1 "/"heternzygotesby intercrossing the chimera with ICR or C57BL/6J strains. ET-1 + homozygoleswere derived flora intercrosses of heterozygctes. ET-I" hetemzygotes appear normal and were fertile. However, All the ET-1 + homozygc4es, in which no ET-1 mRNAor pepfide was de~-t~d, died at birth because of inability to breathe. All the homozygotes represented conspicuous craniofacial anomaly including micrognathia, microglussia, microtia, cleft palate, hypoplasia of submandibular gland and abnormality in thyroid cartilage and hyoid. At the early stage of organogenesis, pharyngeal arch development including Meckel'sand Reichert's cartilage formation was severely impaired in ET-1 + homozygctes. Organ culture of the mandibular arches showed thai the formation of tongue epithelium is poor in ET-1 + homozygotesand is markedly improved by a supplement of 10 • M ET-1. Furthermore, local expression of ET-1 gene in the pharyngeal arches was revealed by/n situ hybridization. In hcterozygotes, the ET-1 levels in the lung (70.0 ± 7.5 pg/mg protein) and plasma (1.1 ± 0.2 pg/ml) were decreased in comparison to those in wild-type mice (lung, 153.1 ± 12.6; plasma, 1.8±0.3). In heterozygous and wild-type llttormates, blood pressure and heart rate were measured through a catheter placed in
the femoral artery under conscious and unrestrained condition. The data were as follows: ~P(n~ MB~n~ DB~mmHg~ ~ ) Wild-Type(n=9)
122 ± 2
105 ~- 2
88 ~- 3
596 ± 26
Hetero(n=13)
133 ± 2 * 1 1 6 * 2 * 9 9 :t: 2 * 5 9 1 ± 14 *p<0.01 Unexpectedly, blood pressure was mildly but significantly increased in heternzygotes in comparison to wild-type mice. The pressor effects of
exogenous ET-1 and NO synthase inhibitor, L-NMMA,were not different betweea ET-1 '/" heterozygotes and wild-type nli~, suggesting that hypersensitivity to El'-1 or decreased production of NO cannot explain the blood pressure elevation in El'-1" heterozygotes. Although the precise mechanisms remains unclear, these results suggest that ET- 1 is essential for normal embryonic developmentand may also play a role in the regulation of blood pressure as a depressor rather than as a pressor.
109
K.Yokokawa,
MODULATES THE
K.Yasunari,
K.Murakawa,
MITOGENIC
H.Kano,
EFFECT
M.Ikeda,
OF PDGF
M.Minami,
ON
T.Hanehira,
First Department of Internal Medicine, Osaka City University Medical School, Osaka 545, JAPAN We have previously shown that cultured rat mesangial cells secrete endothelin-1 and this secretion is stimulated by PDGF (Am J Physiol 266:E673,1993). Here, we examined whether PDGF stimulate mesangial cell proliferation through the modulation of the endogenous production of endothelin-1 in these cells. Endothelin-1 level was measured by radioimmunoassay. Mitogenesis experiments were assessed by tritiated thymidine incorporation into DNA under highest concentrations (10ng/ml) of PDGF in the presence and absence of antiendothelin-1 antiserum or a selective A-type endotbelin receptor (ETA receptor) antagonist, BQ-123. PDGF AB and BB significantly stimulated mesangial cell production of endothelin-1, but PDGF AA did not affect endothelin-1 production. PDGF AB and BB potently stimulate thymidine incorporation, but PDGF AA did not affect thymidine incorporation. This stimulation was significantly attenuated in the presence of either anti-endothelin-1 antiserum or BQ-123 (10-7-10 -5 M). PDGF AB and BB significantly stimulate cell number and this stimulation was also attenuated in the presence of either anti-endothelin-1 antiserum or BQ-123. Anti-endothelin-1 antiserum and the highest concentration (10-SM) of BQ-123 did not affect basal thymidine incorporation and cell number significantly. Results suggest that PDGF AB and BB stimulate endothelin-1 secretion in rat mesangial cells through PDGF t3receptors, and endogenously produced endothelin-1 serves to modulate the effect of PDGF AB and BB, probably via ETA receptors in these cells. These data raise the possibility of the renoprotective effect of ETA receptor blocking in a certain renal disease.
$6-A-1-02 TARGETED DISRUPTIONOF THE MOUSEENDOTHELIN-1GENE I-L Knrihar#, Y. Knrihar#, K SuzukPa, T. Kodama~, K. Maemnra1,1~ NagaP, K Oda', T. KuwakP, T. Ishikawa~, M. Kumada', Y. YazakiI 1)The Third Department of Internal Medicine,3)Department of Pathology, 4)Department of Physiology, University of Tokyo, Tokyo, Japan 2)Chugai Pharmaceutical Co. Ltd., Shiznoka, Japan To elucidate the pathophysiological rain of endothelin-1 (ET-1), we established ET-1 knockout mice by gene targeting and analyzed the phenotype of homozygotes and hetero~ygotes. We introduced the targeting vector constructed from 7.5 kb mouse ET- 1 genornicDNA fragment into A3.1 ES cells by electtopomfion, and obtained 35 targeted ES clones using positive-negative selection. One of the targeted clones gave two chimeric mice with germ-line transmission and we obtained ET-1 "/"heternzygotesby intercrossing the chimera with ICR or C57BL/6J strains. ET-1 + homozygoleswere derived flora intercrosses of heterozygctes. ET-I" hetemzygotes appear normal and were fertile. However, All the ET-1 + homozygc4es, in which no ET-1 mRNAor pepfide was de~-t~d, died at birth because of inability to breathe. All the homozygotes represented conspicuous craniofacial anomaly including micrognathia, microglussia, microtia, cleft palate, hypoplasia of submandibular gland and abnormality in thyroid cartilage and hyoid. At the early stage of organogenesis, pharyngeal arch development including Meckel'sand Reichert's cartilage formation was severely impaired in ET-1 + homozygctes. Organ culture of the mandibular arches showed thai the formation of tongue epithelium is poor in ET-1 + homozygotesand is markedly improved by a supplement of 10 • M ET-1. Furthermore, local expression of ET-1 gene in the pharyngeal arches was revealed by/n situ hybridization. In hcterozygotes, the ET-1 levels in the lung (70.0 ± 7.5 pg/mg protein) and plasma (1.1 ± 0.2 pg/ml) were decreased in comparison to those in wild-type mice (lung, 153.1 ± 12.6; plasma, 1.8±0.3). In heterozygous and wild-type llttormates, blood pressure and heart rate were measured through a catheter placed in
the femoral artery under conscious and unrestrained condition. The data were as follows: ~P(n~ MB~n~ DB~mmHg~ ~ ) Wild-Type(n=9)
122 ± 2
105 ~- 2
88 ~- 3
596 ± 26
Hetero(n=13)
133 ± 2 * 1 1 6 * 2 * 9 9 :t: 2 * 5 9 1 ± 14 *p<0.01 Unexpectedly, blood pressure was mildly but significantly increased in heternzygotes in comparison to wild-type mice. The pressor effects of
exogenous ET-1 and NO synthase inhibitor, L-NMMA,were not different betweea ET-1 '/" heterozygotes and wild-type nli~, suggesting that hypersensitivity to El'-1 or decreased production of NO cannot explain the blood pressure elevation in El'-1" heterozygotes. Although the precise mechanisms remains unclear, these results suggest that ET- 1 is essential for normal embryonic developmentand may also play a role in the regulation of blood pressure as a depressor rather than as a pressor.
109